TIP aquaporins in Cyperus esculentus: genome-wide identification, expression profiles, subcellular localizations, and interaction patterns

Background Tonoplast intrinsic proteins (TIPs), which typically mediate water transport across vacuolar membranes, play an essential role in plant growth, development, and stress responses. However, their characterization in tigernut (Cyperus esculentus L.), an oil-bearing tuber plant of the Cyperaceae family, is still in the infancy. Results In this study, a first genome-wide characterization of the TIP subfamily was conducted in tigernut, resulting in ten members representing five previously defined phylogenetic groups, i.e., TIP1–5. Although the gene amounts are equal to that present in two model plants Arabidopsis and rice, the group composition and/or evolution pattern were shown to be different. Except for CeTIP1;3 that has no counterpart in both Arabidopsis and rice, complex orthologous relationships of 1:1, 1:2, 1:3, 2:1, and 2:2 were observed. Expansion of the CeTIP subfamily was contributed by whole-genome duplication (WGD), transposed, and dispersed duplications. In contrast to the recent WGD-derivation of CeTIP3;1/-3;2, synteny analyses indicated that TIP4 and − 5 are old WGD repeats of TIP2, appearing sometime before monocot-eudicot divergence. Expression analysis revealed that CeTIP genes exhibit diverse expression profiles and are subjected to developmental and diurnal fluctuation regulation. Moreover, when transiently overexpressed in tobacco leaves, CeTIP1;1 was shown to locate in the vacuolar membrane and function in homo/heteromultimer, whereas CeTIP2;1 is located in the cell membrane and only function in heteromultimer. Interestingly, CeTIP1;1 could mediate the tonoplast-localization of CeTIP2;1 via protein interaction, implying complex regulatory patterns. Conclusions Our findings provide a global view of CeTIP genes, which provide valuable information for further functional analysis and genetic improvement through manipulating key members in tigernut. Supplementary Information The online version contains supplementary material available at 10.1186/s12870-024-04969-x.


Background
Aquaporins (AQPs), which belong to the ancient major intrinsic protein (MIP) superfamily, constitute a family of small integral membrane proteins facilitating the fast and passive movement of water and other small solutes [1].In higher plants, AQPs could be classified into five subfamilies on the basis of sequence similarity, i.e., plasma membrane intrinsic protein (PIP), tonoplast intrinsic protein (TIP), NOD26-like intrinsic protein (NIP), small basic intrinsic protein (SIP), and X intrinsic protein (XIP) [2][3][4].Among them, PIPs and TIPs, which are typically located in cell and vacuolar membranes, respectively, are vital for maintaining proper cytosolic osmolarity in plant cells [5].In contrast to only two phylogenetic groups present in the PIP subfamily, the TIP subfamily is highly diverse and five groups have been described not only in Arabidopsis (Arabidopsis thaliana (L.) Heynh) but also in rice (Oryza sativa L.), two model plants for eudicots and monocots, respectively, implying their early origin [6,7].Interestingly, despite the presence of ten members in both Arabidopsis and rice, their group distribution is distinct, reflecting the occurrence of three independent whole-genome duplication (WGD) events after monocot-eudicot divergence [8,9].Functional analyses of TIP genes have been performed in a wide range of plant species, e.g., Arabidopsis, maize (Zea mays L.), rice, wheat (Triticum aestivum L.), and tobacco (Nicotiana tabacum L.) [10][11][12][13][14][15][16][17].In addition to water, TIPs have also been shown to transport urea, ammonia (NH 3 ), hydrogen peroxide (H 2 O 2 ), and glycerol, corresponding to diverse aromatic/arginine (ar/R) selectivity filters that determine the substrate specificity [10][11][12][13][14][15][16][17].The crystal structure of Arabidopsis AtTIP2;1, a water and ammonia transporter, was resolved in 2016 [18].Compared with other AQPs such as SoPIP2;1 of spinach (Spinacia oleracea L.) [19], the side chain of the conserved R residue in AtTIP2;1 is pushed to the side of the pore by an H residue located in loop C (LC), appearing as a fifth residue of an extended selectivity filter and the relatively wide pore and the polar nature of the selectivity filter allowing the ammonia permeability [18].Tigernut (Cyperus esculentus L. var.sativus) is an herbaceous perennial C 4 plant of the Cyperaceae family within the Poales order, which also includes the wellknown Poaceae family [20][21][22][23].Although most likely originating from the Mediterranean and Southwest Asia, tigernut is now widely distributed in tropical, subtropical, temperate, and even cold zones [21,24,25].It has emerged as a novel oil crop that uniquely accumulates up to 35% of oil in underground tubers [26][27][28].Despite its strong anti-adversity and wide adaptability [24], the mechanism underlying is poorly understood.Given essential roles of TIPs in plant growth, development, and stress responses [17], in the current study, we took advantage of recently available genome and transcriptome data [29,30] to identify the complete set of TIP subfamily genes.Their gene localizations, gene structures, sequence characteristics, and evolutionary relationships were comprehensively examined and compared with that of Arabidopsis and rice, which have extensively studied [7,10,14].Moreover, gene expression profiles, protein subcellular localizations, and interaction patterns were also investigated, which facilitate further functional analysis and application.

Identification and classification of TIP genes in tigernut
As shown in Table 1, a total of ten TIP genes were identified from the tigernut genome and all of them were predicted by the genome annotation [30] and detected in the full-length transcriptome [28], supporting their expression.To uncover their evolutionary relationships, an unrooted phylogenetic tree from deduced polypeptides of CeTIP, OsTIP, and AtTIP genes was constructed as shown in Fig. 1a, which clusters ten CeTIP genes into five main groups, i.e., three TIP1s, three TIP2s, two TIP3s, one TIP4, and one TIP5.Among them, TIP1 and − 2 could be divided into several subgroups.Surprisingly, the group distribution in tigernut appears be more similar to Arabidopsis but distinct from rice, though tigernut and rice share a closer biological relationship and both are monocots belonging to Poales [20].

Analyses of exon-intron organization and conserved motifs
To gain insights into structural divergence of CeTIP genes, their gene structures and conserved motifs of deduced proteins were further investigated and compared with that of rice and Arabidopsis.As shown in Fig. 1b, all CeTIP genes possess two introns and three exons, by contrast, nearly half of TIP genes in rice and Arabidopsis harbor a single intron and AtTIP1;3 is intronless.Genes with less than two introns were found in TIP1 and − 4 of rice and TIP1 and − 2 of Arabidopsis, respectively (Fig. 1b).Without any exception, all CeTIP genes feature the typical GT/AG splice junction.Generally, intron 1 is located near the end of transmembrane helix 1 (TM1), whereas intron 2 is located just before the A/R/E/S/P/G/H codon of the LC.Whereas intron 1 is the phase 1 intron that is located within codons, intron 2 is usually the phase 0 intron that is located between codons (Fig. 1b).Nevertheless, intron 2 of both CeTIP1;1 and − 1;3 exhibits the phase 1 intron (Fig. 1b), which was shown to locate at the C-terminus of TM4 and the loop D (LD), respectively (Additional file 4).Fifteen motifs identified using MEME are shown in Fig. 1c.Only Motifs 2 and 3, which are characterized as TM1 and LB-TM3 (Additional file 4), are shared by all 30 TIPs examined.Motifs 1 and 4-7 are also broadly present: Motif 1, which is characterized as TM5-LE-TM6, is replaced by Motif 8 in OsTIP4;1 and all TIP5s; Motif 4, which is characterized as LC-TM4-LD, is absent from OsTIP3;2 and OsTIP4;3 and replaced by Motif 9 in all TIP5s; Motif 5, which is characterized as LA-TM2, is absent from AtTIP4;1, OsTIP4;1, OsTIP4;2, and AtTIP5;1; Motif 5, which is characterized as a part of TM6, is absent from CeTIP5;1 and OsTIP5;1; Motif 5, which is located at the C-terminus, is absent from CeTIP3;2, CeTIP4;1, AtTIP2;2, AtTIP2;3, AtTIP5;1, and OsTIP5;1 and replaced by Motif 14 in CeTIP2;2, OsTIP2;1, OsTIP4;2, and CeTIP5;1.Motifs 10, 11, and 15, which are located at the N-terminus, are mainly present in TIP1s, TIP2s/TIP4s, and AtTIP3s, respectively.Motif 12, which is located at LC, is restricted to TIP3 and replaced by Motif 13 in TIP4s, AtTIP2;1, AtTIP2;2, AtTIP2;3, CeTIP2;3, and AtTIP1;1 (Fig. 1c and Additional file 4).It's worth noting that the motif composition in TIP5s is obviously different from other groups, reflecting their sequence divergence.Nevertheless, all of them harbor one conserved MIP domain that includes six typical TMs (TM1-6), two half helixes (HB and HE), and two typical NPA motifs as observed in SoPIP2;1 [19], though the ar/R selectivity filter is variable (Fig. 1d).Among them, CeTIP1;3 exhibits the H-I-A-A variant in contrast to the H-I-A-V filter widely present in TIP1.TIP3 and − 4 usually feature the H-I-A-R filter, however, CeTIP3;1 exhibits the H-T-A-R variant that is not observed in other members.Whereas all TIP2s feature the H-I-G-R filter, CeTIP5;1 exhibits the Q-V-G-R filter that is different from that of both OsTIP5;1 (Q-V-A-R) and AtTIP5;1 (N-V-G-C).An L residue, corresponding to the L 197 of SoPIP2;1 that was proven to be involved in gating [19], is only present in CeTIP4;1, OsTIP4;3, and OsTIP5;1.In most cases, this position is replaced by an I residue.An H residue, corresponding to the H 197 of AtPIP2;2 that was reported to undergo gating by cytosolic pH via protonation [33], is found in CeTIP3;1 and OsTIP3;1.Interestingly, an H residue, corresponding to the H 131 in the LC of AtTIP2;1 and VvTIP2;1 that is essential for ammonia permeability [18] and involved in gating by cytosolic pH [34], was found not only in TIP2s but also TIP4s, implying their similar functions.

Global expression profiles of CeTIP genes
To reveal the global expression profiles of CeTIP genes, RNA-seq data for nine tissues/developmental stages with three biological repeats each were first examined, i.e., two stages of developmental leaf (young and mature), sheath of mature leaf, root, rhizome, shoot apex, and three stages of developmental tuber (40, 80, and 120 days after sowing (DAS)).As shown in Fig. 3a, the total CeTIP transcripts were most abundant in roots, followed by rhizomes, moderate in sheaths, young leaves, and tubers of 120 and 80 DAS, and relatively low in mature leaves, tubers of 40 DAS, and shoot apexes.Interestingly, except for roots and tubers of 40 DAS, CeTIP1;1 and − 2;1 represent two dominant members in all other samples examined, contributing 82.46-95.72% of total TIP transcripts.In roots, CeTIP2;2 and − 2;3 were shown to express more than − 1;1 and − 2;1, and these four members constituted more than 91.89% of total TIP transcripts.Additionally, CeTIP1;2 and − 4;1 were also abundant in roots, whose transcripts were shown to be comparative or considerably more than that of most other samples.In shoot apexes, CeTIP4;1 represented the third most expressed member, contributing 15.02% of total TIP transcripts.By contrast, this gene was rarely expressed in leaves and sheaths.Interestingly, CeTIP3;1 appeared to be tuber-specific and highly abundant in tubers of 120 DAS, whereas CeTIP3;2 was lowly expressed in all samples examined.

Expression profiles of CeTIP genes during leaf development and diurnal fluctuation
As shown in Fig. 3a, most CeTIP genes were significantly down-regulated in mature leaves relative to young leaves, including two dominant genes CeTIP1;1 and − 2;1.However, this is the case under a natural condition.To provide a more accurate determination, plants grown in a greenhouse as described before [23] were adopted and three representative stages of developmental leaf (i.e.young, mature, and senescing) were subjected to qRT-PCR analysis.As shown in Fig. 3b, both CeTIP1;1 and − 2;1 exhibited a bell-shaped expression pattern, peaking in mature leaves, though the difference of CeTIP1;1 between young and mature leaves was not significant.
Diurnal fluctuation expression patterns of CeTIP1;1 and − 2;1 in mature leaves were further examined.As shown in Fig. 3c, since the onset of light at 8 h, their transcripts usually increased along with the extension of the light time, peaking at 16 h and 12 h, respectively, though a slight drop was observed at 12 h for CeTIP1;1.Whereas CeTIP2;1 was expressed more in light (i.e. 8 h, 12 h, 16 h, and 20 h) relative to dark (i.e.24 h and 4 h), the transcripts of CeTIP1;1 at 8 h and 12 h were significantly lower than that in dark (Fig. 3c).

Expression profiles of CeTIP genes during tuber development
The oil-bearing tubers are derived from rhizomes and the development process includes three main stages, i.e., initiation, swelling, and maturation, spanning approximately 35 d [22].In contrast to gradual increase of dry weight and oil during tuber development, the water content usually maintains up to 85.0% until a significant drop to less than 45.0% at the stage of maturation [22,26,28], implying a key role of water for tuber development and metabolism.Among three swelling stages profiled, except for CeTIP1;3 that peaked at 40 DAS, transcripts of CeTIP2;1 and − 3;1 were shown to gradually increase, whereas five genes (i.e.CeTIP1;1, -1;2, -2;2, -2;3, and − 4;1) exhibited an apparent unimodal expression pattern, peaking at 80 DAS (Fig. 3a).
For qRT-PCR analysis, seven stages were examined, i.e., 1, 5, 10, 15, 20, 25, and 35 days after tuber initiation (DAI), representing initiation, five stages of swelling, and maturation as described before [22].As shown in Fig. 3d, CeTIP1;1 exhibited a nearly bell-shaped expression pattern, peaking at 15 DAI, which is similar to the transcriptome profiling.By contrast, a bimodal expression pattern was observed for CeTIP2;1, peaking at 10 and 25 DAI, respectively.It's worth noting that, for both genes, transcripts at the maturation stage were significantly lower than any other stage (Fig. 3d), corresponding to relatively low water content at this stage [22].

Subcellular localization analysis
According to bioinformatic prediction using Plant-mPLoc, all CeTIP proteins were shown to localize to the tonoplast.To verify the result, two dominant members CeTIP1;1 and − 2;1 were selected for experimental confirmation.As shown in Fig. 4, green fluorescence signals of CeTIP1;1-GFP were widely detected in vacuoles of tobacco leaves, highly coinciding with the tonoplast marker AtTIP1;1-RFP [10].By contrast, to our surprise, fluorescence signals of CeTIP2;1-GFP were only found in cell membranes, coinciding with the plasma membrane marker HbPIP2;3-RFP [35].

Protein interaction patterns
Constitutive expression and high abundance of CeTIP1;1 and − 2;1 (Fig. 3a) suggest that their encoded proteins may interact with each other.To confirm the hypothesis, bimolecular fluorescence complementation (BiFC) analysis was conducted.As shown in Fig. 5, in contrast to no signals were detected for four negative controls, green fluorescence signals were found in vacuoles of tobacco leaves co-transformed with Ecn-CeTIP1;1 and Enn-CeTIP2;1, supporting their interaction.Moreover, homologous interaction was also observed for CeTIP1;1 but not − 2;1.These results imply that CeTIP1;1 is more likely to function in homo and heteromultimer, whereas CeTIP2;1 functions in heteromultimer (Fig. 5).

The tigernut genome encodes ten TIP genes with two introns
Since the first TIP gene (i.e.AtTIP1;1 orγ-TIP1) was characterized in Arabidopsis [10], its homologs have been identified in a high number of plant species, varying from eight to more than 23 members in soybean (Glycine max (L.) Merr.) [10,16,17,36,37].In the present study, a number of ten TIP genes that is equal to that of Arabidopsis were identified from tigernut, an oil-bearing tuber plant of the Cyperaceae family, which represents a sister family to Poaceae within the Poales order [20].The CeTIP gene amounts are comparative to or relatively less than 9-15 members present in most Poaceae plants, e.g., rice, barley (Hordeum vulgare L.), Brachypodium distachyon (L.) P. Beauv., foxtail millet (Setaria italic (L.) P. Beauv.), green foxtail (S. viridis (L.) P. Beauv.), sorghum (Sorghum bicolor (L.) Moench), and maize [7,16,[38][39][40], Additional file 3. Nevertheless, all these genes could be assigned into five phylogenetic groups as defined in Arabidopsis, i.e., TIP1-5 [6].Moreover, in contrast to a high number of TIP genes present in Arabidopsis and rice harboring no or a single intron, all CeTIP genes feature two introns.Constant positions of two introns in most TIP genes support independent loss of one or both introns, whereas varied intron phase and locations of intron 2 in CeTIP1;1 and − 1;3 imply the initial loss followed by gain of a new intron.Similar phenomenon was also reported for AtPIP2;4 [6].
Diurnal variation of AQP genes has been reported in several species [7,62].For example, the transcripts of OsTIP1;2 and − 2;1 in roots were shown to exhibit a clear diurnal change with a large amplitude, peaking in light and dropping to the basal level in dark [7].Similar trend was also observed in this study, and transcripts of both CeTIP1;1 and − 2;1 peaked in the daytime, 8 h-4 h after the onset of light, respectively (Fig. 3c).

Distinct subcellular localizations and interaction patterns of CeTIP1;1 and − 2;1
As the name suggests, TIPs are located mostly in tonoplasts of plant cells, which function in tetramers [5,10,18].Interestingly, CeTIP1;1 and − 2;1, two dominant CeTIP genes identified in tigernut, encode proteins with distinct subcellular localizations and interaction patterns.When transiently overexpressed in tobacco leaves, CeTIP1;1 is clearly located in the vacuole membrane, in contrast to CeTIP2;1 that is located in the cell membrane.Moreover, CeTIP2;1 could not interact itself, whereas CeTIP1;1 functions in homo/heteromultimer and could mediate the tonoplast-localization of CeTIP2;1.It's worth noting that proteomic analysis suggests the presence of AtTIPs in the thylakoid membrane of chloroplast [63], and AtTIP3s were shown to localize in both the plasma membrane and tonoplast of maturing and germinating seeds [61].Compared with PIPs, only a few studies have been performed to investigate the interaction pattern of TIPs.In Arabidopsis, AtTIP2;1 could not only interact itself but also interact with AtTIP1;2 and − 3;1 [64].Thereby, the reason for why CeTIP2;1 could not form homomultimer needs to be further studied.

Conclusions
To our knowledge, this is the first genome-wide characterization of the TIP subfamily in tigernut, an oil-bearing tuber plant of the Cyperaceae family.Ten members representing five phylogenetic groups identified in tigernut are equal to that present in two model plants Arabidopsis and rice, however, the group composition and/or evolution pattern are different and complex orthologous relationships were observed.Expansion of the CeTIP subfamily was contributed by WGD, transposed, and dispersed duplications.Whereas CeTIP3;1/-3;2 are recent WGD repeats, TIP4 and − 5 were proven to be old WGD repeats of TIP2, appearing sometime before monocoteudicot divergence.CeTIP genes exhibit diverse expression profiles and are subjected to developmental and diurnal fluctuation regulation.Moreover, CeTIP1;1 and − 2;1, two dominant members, exhibit distinct subcellular localizations and interaction patterns.These findings provide valuable information for further functional analysis and genetic improvement through manipulating key members such as CeTIP1;1 and − 2;1 in tigernut.

Synteny analysis and characterization of orthologs
Synteny analysis was conducted as previously described [28,65], where duplicate pairs were identified using the all-to-all BLASTP method and syntenic blocks were inferred using MCScanX implemented in TBtools-II [66]: E-value, 1e-10; BLAST hits, 5. Orthologs between different species were identified using the BRH method and information from synteny analysis, whereas different modes of gene duplication were identified using the Dup-Gen_finder pipeline [43].

Plant materials
For gene cloning and expression analysis, the tigernut variety Reyan3 as described before [23,28] was used, and plants were grown in a greenhouse under the condition of 25 ± 0.5℃, 60-70% relative humidity, 5,000 lx, and a 12 h light/12 h dark cycle.For a day, the period from 8 a.m to 8 p.m was set to light, and mature leaves with dark green in appearance were sampled every four hours from the onset of light.Three representative stages of developmental leaf, i.e., young, mature, and senescing, were also collected at 8 a.m, where the chlorophyll content of young and senescing leaves are just a half relative to mature leaves.As for representative stages of developmental tuber, fresh tubers at 1, 5, 10, 15, 20, 25, and 35 DAI were collected as described before [22].All samples with three biological replicates were quickly freezed with liquid nitrogen and stored at -80℃.For subcellular localization and BiFC analyses, N. benthamiana L. plants were also grown in the greenhouse as described above.

Gene expression analysis
Global expression profiles of CeTIP genes were investigated using Illumina RNA-seq samples as shown in Additional file 5, which are paired reads with 150 bp.Raw reads in the FASTQ format were obtained using fastq-dump, and quality control was performed using Trimmomatic [69].Read mapping was performed using HISAT2 (v2.2.1) [70] with default parameters and relative transcript levels were presented in FPKM (Fragments per kilobase of exon per million fragments mapped).Differential expression genes were identified using DESeq2 (v1.22.1)[71] with parameters of "log 2 Fold Change > = 1" and FDR < 0.05.For qRT-PCR analysis, total RNAs were extracted using the RNAprep Pure Plant Kit (Tiangen Biotech Co., Beijing, China); synthesis of the first-strand cDNA from high quality RNAs was carried out using the PrimeScript® RT reagent kit with gDNA Eraser (Takara, Dalian, China); and PCR reaction was carried out as described before [23].Primers used in this study are shown in Additional file 6, where CeUCE2 and CeTIP41 were used as two reference genes.The relative expression level of target genes was normalized using the 2 −ΔΔCt method and statistical significance was performed using SPSS Statistics 20, where difference significance was tested following Duncan's one-way multiple-range post hoc ANOVA.

Fig. 1
Fig. 1 Phylogenetic analysis and structural features of the TIP subfamily in tigernut, rice and Arabidopsis.a Shown is an unrooted phylogenetic tree resulting from full-length TIPs with MEGA6 (maximum likelihood method and bootstrap of 1,000 replicates), where the distance scale denotes the number of amino acid substitutions per site and the name of each group is indicated next to the corresponding clade.b Shown are the exon-intron structures.0 and 1 indicate intron phases.c Shown is the distribution of conserved motifs among TIPs, where different motifs are represented by different color blocks as indicated and the same color block in different proteins indicates a certain motif.d Shown are the conserved residues such as dual NPA motifs and Ar/R selectivity filter.Unusual residues are marked in red.(At Arabidopsis thaliana, Ar/R aromatic/arginine, bp base pair, Ce Cyperus esculentus, H2 transmembrane helix 2, H5 transmembrane helix 5, LB loop B, LE loop E, NPA Asn-Pro-Ala, Os Oryza sativa, TIP tonoplast intrinsic protein)

Fig. 2
Fig. 2 Duplication events of CeTIP genes and synteny analysis within and between tigernut, rice, and Arabidopsis.a Duplication events detected in tigernut.Serial numbers are indicated at the top of each scaffold, and the scale is in Mb.Duplicate pairs identified in this study are connected using lines in different colors, i.e., tandem (shown in green), transposed (shown in purple), dispersed (shown in gold), and WGD (shown in red).b Synteny analysis within and between tigernut, rice, and Arabidopsis.Shown are TIP-encoding chromosomes/scaffolds (tigernut, pink; rice, dark green; Arabidopsis, bluishgrey) and only syntenic blocks that contain TIP genes are marked (intra-species, red; inter-species, purple).(At Arabidopsis thaliana, Ce Cyperus esculentus, Mb megabase, Os Oryza sativa, Scf scaffold, TIP tonoplast intrinsic protein, WGD whole-genome duplication)

Fig. 3
Fig. 3 Expression profiles of CeTIP genes.a Tissue-specific expression profiles of CeTIP genes.DAS40, DAS80, and DAS120 represent tubers of 40, 80 and 120 DAS.Color scale represents FKPM normalized log 2 transformed counts where blue and red indicate low and high expression, respectively.b Expression profiles of CeTIP1;1 and − 2;1 during leaf development.c Diurnal fluctuation expression patterns of CeTIP1;1 and − 2;1 in mature leaf.d Expression profiles of CeTIP1;1 and − 2;1 during tuber development.For qRT-PCR analysis, CeUCE2 and CeTIP41 were used as two reference genes.Bars and error bars indicate the mean ± SD (n = 3) and uppercase letters indicate difference significance tested following Duncan's one-way multiple-range post hoc ANOVA (P < 0.01).(Ce Cyperus esculentus, DAI days after tuber initiation, DAS days after sowing, TIP tonoplast intrinsic protein)

Table 1
List of ten TIP genes identified in tigernut.(AA amino acid, AI aliphatic index, At Arabidopsis thaliana, Scf scaffold, GRAVY grand average of hydropathicity, kDa kilodalton, MIP major intrinsic protein, MW molecular weight, Os Oryza sativa, TIP tonoplast intrinsic protein, TM transmembrane helix, WGD whole-genome duplication)